Summary:A current focus of the laboratory is investigation of the physiological functions of the neuropeptide Tuberoinfundibular Peptide of 39 residues (TIP39) and its receptor, the Parathyroid Hormone 2 (PTH2) receptor. These molecules were discovered in this laboratory several years ago. In previous years of this project we mapped the neuroanatomical distributions of TIP39 and the PTH2 receptor. TIP39 is synthesized by three discrete groups of neurons, two at the caudal border of the thalamus and one in the brainstem. TIP39 synthesizing neurons project to several brain areas that are involved in the regulation of emotional function. These areas contain a matching distribution of the PTH2 receptor, and neurons in these regions project to the areas containing TIP39 neurons. Thus the system is ideally positioned to coordinate and modulate functions relevant to mental disorders. Following this anatomical mapping, laboratory projects turned to investigation of hypotheses derived from the distribution of TIP39 and the PTH2R. We found that TIP39 modulates activation of neurons in the paraventricular nucleus of the hypothalamus, which controls several neuroendocrine functions, including release of glucocorticoid stress hormone from the adrenal gland. TIP39 increased the stress hormone corticosterone. TIP39 does this by acting on the terminals of neurons within the paraventricular nucleus that release the classic fast-acting transmitter glutamate, thus it modulates excitatory inputs to neuroendocrine cells. We also found that TIP39 modulates pain sensitivity, acting primarily within the brain to affect the processing of painful sensory information. The brain areas where TIP39 appears to modulate pain are also implicated in affective disorders, so a hypothesis we plan to examine in the future is that TIP39 signaling is involved in the strong relationship between chronic pain and depression. In previous years of the project we found that mice with genetic deletion of the gene encoding TIP39 (TIP39-KO) have increased anxiety-like behavior that depends upon the level of stress created by the testing conditions. Under conditions of minimal stress loss of TIP39 had little effect, while under increased stress animals without TIP39 exhibited significantly greater anxiety-like behavior than mice with normal TIP39 function. We also investigated the role of TIP39 in modulating the effects of stress on cognitive function. Mice lacking TIP39 signaling because of either ligand or receptor loss or acute receptor blockade showed impaired performance in behavioral tests that depend on memory function (object recognition and social recognition tests and Y-maze test), under conditions of novelty-induced arousal but not when acclimated to the testing environment. We also found that TIP39 signaling modulates long-term emotional memory. In a mouse model of post-traumatic stress disorder in which the animals are exposed to a single traumatic event (electric foot-shock) after which fear memory is evaluated by re-exposing them to the context of the traumatic event and measuring the time spent motionless (freezing, a rodent fear-like response) the lack of TIP39 signaling did not cause a detectable change in fear memory one week after the shock. However, both mice lacking the peptide gene as well as mice with null mutation in the PTH2-R gene exhibited greater fear-like behavior than wild-type mice two weeks following the shock. Thus our data suggest that TIP39 signaling may normally limit the detrimental effects of environmental stress on cognitive performance and emotional state. Patients with a number of mental health disorders exhibit much greater dysfunction in a novel environment and under stress than under familiar conditions. We previously found that the neuroanatomical distributions of TIP39 and the PTH2-R are almost identical in humans and rodents, so this suggests that endogenous TIP39 signaling may be involved in limiting deleterious effects of stress in humans. Most of our studies have been performed in male animals, although we did find a significant sexual dimorphism in the postnatal expression of TIP39 in rats. There is a significant post-pubertal decline in TIP39 in male rats and a significantly smaller decline in females, suggesting involvement in sex specific functions. Previously we found that TIP39 modulates release of the pituitary hormone prolactin in male mice. Prolactin is a major mediator of maternal functions, including milk production. In collaboration with Dr. Arpad Dobolyi of Semmelweis University in Hungary, we also found that TIP39 levels are significantly increased in lactating females, that pup suckling activates the neurons that synthesize TIP39, and that acute block of TIP39 signaling inhibits the stimulation of prolactin release normally caused by pup suckling. In a study comparing wild-type and PTH2R-KO mouse dams we found no significant differences in their maternal behaviors, but the pups of PTH2R-KO dams gained weight significantly more slowly than the pups of WT dams in the postnatal period. By mating wild-type males with KO females and KO males with WT females all pups had identical genotypes. Pup weights were the same at birth, indicating that postnatal and not prenatal factors affected the weight gain, which is consistent with TIP39 modulation of the sensitivity of prolactin release to pup suckling. Along with previous data this suggests that TIP39 helps to control which or how effectively hypothalamic excitatory pathways control neuroendocrine function, contributing to an appropriate hormonal response to changing conditions. During this review period we extended our examination of TIP39 regulation of hypothalamic function by investigating its contribution to thermoregulation. This was based on a relatively high level of TIP39 and PTH2R expression in the median preoptic area, a region that has a key role in brain control of body temperature. We found that although mice lacking TIP39 signaling had a normal body temperature and normal daily variation in body temperature when maintained at the vivarium temperature to which they were acclimated, they were severely impaired in heat production in response to acute exposure to a cold environment. Based on PTH2R localization on terminals of glutamatergic neurons within the median preoptic area the data suggest that TIP39 signaling is necessary for a large release of glutamate from these terminals and robust activation of downstream circuits. This type of regulation of some excitatory synapses may be a model for TIP39 effects in other brain areas and underlie its role in stress responses. Thus our working hypothesis is that TIP39 may significantly and dynamically boost the gain in specific stress associated circuits.